Of Keeping and Tipping the Balance: Host Regulation and Viral Modulation of IRF3-Dependent IFNB1 Expression

The type I interferon (IFN) response is a principal component of our immune system that allows to counter a viral attack immediately upon viral entry into host cells. Upon engagement of aberrantly localised nucleic acids, germline-encoded pattern recognition receptors convey their find via a signalling cascade to prompt kinase-mediated activation of a specific set of five transcription factors. Within the nucleus, the coordinated interaction of these dimeric transcription factors with coactivators and the basal RNA transcription machinery is required to access the gene encoding the type I IFN IFNβ (IFNB1). Virus-induced release of IFNβ then induces the antiviral state of the system and mediates further mechanisms for defence. Due to its key role during the induction of the initial IFN response, the activity of the transcription factor interferon regulatory factor 3 (IRF3) is tightly regulated by the host and fiercely targeted by viral proteins at all conceivable levels. In this review, we will revisit the steps enabling the trans-activating potential of IRF3 after its activation and the subsequent assembly of the multi-protein complex at the IFNβ enhancer that controls gene expression. Further, we will inspect the regulatory mechanisms of these steps imposed by the host cell and present the manifold strategies viruses have evolved to intervene with IFNβ transcription downstream of IRF3 activation in order to secure establishment of a productive infection

Analysis of the Fibroblast Growth Factor System Reveals Alterations in a Mouse Model of Spinal Muscular Atrophy

The monogenetic disease Spinal Muscular Atrophy (SMA) is characterized by a progressive loss of motoneurons leading to muscle weakness and atrophy due to severe reduction of the Survival of Motoneuron (SMN) protein. Several models of SMA show deficits in neurite outgrowth and maintenance of neuromuscular junction (NMJ) structure. Survival of motoneurons, axonal outgrowth and formation of NMJ is controlled by neurotrophic factors such as the Fibroblast Growth Factor (FGF) system. Besides their classical role as extracellular ligands, some FGFs exert also intracellular functions controlling neuronal differentiation. We have previously shown that intracellular FGF-2 binds to SMN and regulates the number of a subtype of nuclear bodies which are reduced in SMA patients. In the light of these findings, we systematically analyzed the FGF-system comprising five canonical receptors and 22 ligands in a severe mouse model of SMA. In this study, we demonstrate widespread alterations of the FGF-system in both muscle and spinal cord. Importantly, FGF-receptor 1 is upregulated in spinal cord at a pre-symptomatic stage as well as in a mouse motoneuron-like cell-line NSC34 based model of SMA. Consistent with that, phosphorylations of FGFR-downstream targets Akt and ERK are increased. Moreover, ERK hyper-phosphorylation is functionally linked to FGFR-1 as revealed by receptor inhibition experiments. Our study shows that the FGF system is dysregulated at an early stage in SMA and may contribute to the SMA pathogenesis

Föräldraskap: förändras det när föräldrar mår dåligt?

I denna uppsats undersöks relationen mellan tilltro till föräldraförmåga och föräldrabeteenden över tid och huruvida föräldrars depression och föräldrastress modererar relationen. Tidigare forskning har konstaterat att tilltro till föräldraförmåga är en stark prediktor för föräldrabeteenden, och att föräldrars depression och föräldrastress minskar positiva och ökar negativa föräldrabeteenden. Syftet med uppsatsen är att undersöka om föräldrars välmående är en omständighet då tilltro till föräldraförmåga inte är en lika stark prediktor för positiva föräldrabeteenden. Studiens hypotes är att högre nivåer av föräldrars depression och föräldrastress försvagar associationen mellan tilltro till föräldraförmåga och positiva föräldrabeteenden över tid. Urvalet i studien kommer från ett större projekt som undersökt föräldrars användning av internet och sociala medier, och hur det associerade till föräldraskap. I denna uppsats inkluderas föräldrar (N = 247) till barn i åldrarna 0-5 år och datan är insamlad vid två tillfällen via enkäter. Resultatet av moderationsanalysen går i linje med tidigare forskning och visade en predicerande effekt av tilltro till föräldraförmåga på positiva föräldrabeteenden över ett års tid. Den aktuella studien adderar kunskap om det longitudinella sambandet mellan tilltro till föräldraförmåga och positiva föräldrabeteenden hos föräldrar med barn under sex år. Resultatet visade inga signifikanta modererande effekter av föräldrars depression eller föräldrastress i associationen. Resultaten indikerar att föräldrars välmående inte leder till förändringar i deras föräldrabeteenden över tid, oavsett tilltro till föräldraförmåga. Framtida forskning bör fortsätta undersöka sambandet och använda ett normalfördelat urval gällande föräldrars depression, föräldrastress, tilltro till föräldraförmåga och föräldrabeteenden.This thesis examined the relationship between parental self-efficacy (PSE) and parental behavior, and whether levels of parents' depression or parental stress can moderate the relationship over time. Previous research has inferred that PSE is a strong predictor for parental behaviors, further research suggests that depression and parental stress, decrease positive and increase negative parental behaviors. The purpose of this thesis is to investigate whether parents' well-being, such as parents' depression or parental stress, could be circumstances in which PSE decreases its predicting effect on positive parenting behaviors. The hypothesis is that higher levels of parents' depression or parental stress decreases the strength of the association between PSE and positive parental behavior over time. The sample is drawn from a larger longitudinal study in Sweden that investigated parents' use of internet and social media, and its link to parenting. This study included parents (N = 247) of children 0-5 years old and data was collected at two times via survey responses. Research questions were tested using moderation analysis. Results supported previous findings of a significant predicting effect of PSE on positive parenting behavior over time. In addition, the current study contributes with knowledge that a longitudinal association also exists for parents with children under six years of age. Furthermore, results showed no significant moderating effects of parents' depression or parental stress on the association of PSE and positive parenting behaviors. Findings indicate that levels of parents' depression and parental stress does not change positive parenting behaviors over time, regardless of PSE in this sample. Future research should further investigate parents’ well-being and the effects of PSE and parenting behavior and use a normally distributed sample regarding levels of depression, parental stress, parental self-efficacy and positive parenting behavior

Föräldraskap: förändras det när föräldrar mår dåligt?

I denna uppsats undersöks relationen mellan tilltro till föräldraförmåga och föräldrabeteenden över tid och huruvida föräldrars depression och föräldrastress modererar relationen. Tidigare forskning har konstaterat att tilltro till föräldraförmåga är en stark prediktor för föräldrabeteenden, och att föräldrars depression och föräldrastress minskar positiva och ökar negativa föräldrabeteenden. Syftet med uppsatsen är att undersöka om föräldrars välmående är en omständighet då tilltro till föräldraförmåga inte är en lika stark prediktor för positiva föräldrabeteenden. Studiens hypotes är att högre nivåer av föräldrars depression och föräldrastress försvagar associationen mellan tilltro till föräldraförmåga och positiva föräldrabeteenden över tid. Urvalet i studien kommer från ett större projekt som undersökt föräldrars användning av internet och sociala medier, och hur det associerade till föräldraskap. I denna uppsats inkluderas föräldrar (N = 247) till barn i åldrarna 0-5 år och datan är insamlad vid två tillfällen via enkäter. Resultatet av moderationsanalysen går i linje med tidigare forskning och visade en predicerande effekt av tilltro till föräldraförmåga på positiva föräldrabeteenden över ett års tid. Den aktuella studien adderar kunskap om det longitudinella sambandet mellan tilltro till föräldraförmåga och positiva föräldrabeteenden hos föräldrar med barn under sex år. Resultatet visade inga signifikanta modererande effekter av föräldrars depression eller föräldrastress i associationen. Resultaten indikerar att föräldrars välmående inte leder till förändringar i deras föräldrabeteenden över tid, oavsett tilltro till föräldraförmåga. Framtida forskning bör fortsätta undersöka sambandet och använda ett normalfördelat urval gällande föräldrars depression, föräldrastress, tilltro till föräldraförmåga och föräldrabeteenden.This thesis examined the relationship between parental self-efficacy (PSE) and parental behavior, and whether levels of parents' depression or parental stress can moderate the relationship over time. Previous research has inferred that PSE is a strong predictor for parental behaviors, further research suggests that depression and parental stress, decrease positive and increase negative parental behaviors. The purpose of this thesis is to investigate whether parents' well-being, such as parents' depression or parental stress, could be circumstances in which PSE decreases its predicting effect on positive parenting behaviors. The hypothesis is that higher levels of parents' depression or parental stress decreases the strength of the association between PSE and positive parental behavior over time. The sample is drawn from a larger longitudinal study in Sweden that investigated parents' use of internet and social media, and its link to parenting. This study included parents (N = 247) of children 0-5 years old and data was collected at two times via survey responses. Research questions were tested using moderation analysis. Results supported previous findings of a significant predicting effect of PSE on positive parenting behavior over time. In addition, the current study contributes with knowledge that a longitudinal association also exists for parents with children under six years of age. Furthermore, results showed no significant moderating effects of parents' depression or parental stress on the association of PSE and positive parenting behaviors. Findings indicate that levels of parents' depression and parental stress does not change positive parenting behaviors over time, regardless of PSE in this sample. Future research should further investigate parents’ well-being and the effects of PSE and parenting behavior and use a normally distributed sample regarding levels of depression, parental stress, parental self-efficacy and positive parenting behavior

Of Keeping and Tipping the Balance: Host Regulation and Viral Modulation of IRF3-Dependent Expression.

The type I interferon (IFN) response is a principal component of our immune system that allows to counter a viral attack immediately upon viral entry into host cells. Upon engagement of aberrantly localised nucleic acids, germline-encoded pattern recognition receptors convey their find via a signalling cascade to prompt kinase-mediated activation of a specific set of five transcription factors. Within the nucleus, the coordinated interaction of these dimeric transcription factors with coactivators and the basal RNA transcription machinery is required to access the gene encoding the type I IFN IFNβ (IFNB1). Virus-induced release of IFNβ then induces the antiviral state of the system and mediates further mechanisms for defence. Due to its key role during the induction of the initial IFN response, the activity of the transcription factor interferon regulatory factor 3 (IRF3) is tightly regulated by the host and fiercely targeted by viral proteins at all conceivable levels. In this review, we will revisit the steps enabling the trans-activating potential of IRF3 after its activation and the subsequent assembly of the multi-protein complex at the IFNβ enhancer that controls gene expression. Further, we will inspect the regulatory mechanisms of these steps imposed by the host cell and present the manifold strategies viruses have evolved to intervene with IFNβ transcription downstream of IRF3 activation in order to secure establishment of a productive infection

Crosstalk of neurotrophic factor signaling and ROCK pathway leading to neurite outgrowth – involvement of SMN protein.

<p>FGF-signaling promotes neurite outgrowth by small GTPase-dependent-, ERK- and Akt-pathways. Activation of the small GTPases Rac and Cdc42 and inhibition of the negative effector RhoA promotes outgrowth by posttranslational mechanisms. The ERK and Akt pathways, however, change transcriptional profiles towards an outgrowth promoting state. Both pathways functionally interact with RhoA-downstream-target Rho-kinase (ROCK) as a central node <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031202#pone.0031202-Lin1" target="_blank">[58]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031202#pone.0031202-Ehrenreiter1" target="_blank">[65]</a>. ROCK thereby is important for integration and processing signals of the two major pathways involved in neurite outgrowth. Interestingly, recent findings of our group suggest a role of SMN-Profilin2a interaction in ROCK-pathway dependent outgrowth. SMN-reduction thereby leads to changes in phosphorylation-dependent regulation of ROCK-downstream targets indicated by red arrows <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031202#pone.0031202-Nlle1" target="_blank">[25]</a>. Moreover, SMA-patient derived SMN-S230L-mutation does not interact with Prof2a and acts dominant negative on neurite outgrowth when overexpressed <i>in vitro </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031202#pone.0031202-Nlle1" target="_blank">[25]</a>. In this study, FGFR-1 was found to be upregulated under SMN-knockdown and consistently Akt and ERK showed hyper-phosphorylations. Thereby a sequestration of ROCK via Prof2a binding might enhance ERK-hyper-phosphorylation in a FGFR dependent manner.</p

Expression and regulation of FGFs in spinal cord and NSC34-cells.

<p>(<b>A</b>) Transcript abundances relative to GAPDH in control mice spinal cords and scrambled siRNA transfected NSC34-cells. FGF mRNA levels were measured by qRT-PCR in pooled samples of P5 control mice tissues and scrambled siRNA transfected cells relative to GAPDH as internal control. Transcript abundances were calculated from ΔC_T-values. Unpaired t-tests of technical repetitions of measurements in cell-culture compared to tissue abundances were performed (n = 2; n.s. = non significant, *p<0.05, **p<0.01, ***p<0.001). Bars represent means with standard deviations (SD). (<b>B</b>) Fold-change of FGF transcript levels in SMA-mice spinal cords and NSC34 cells after SMN-knockdown. FGF transcript concentration was measured by qRT-PCR in SMA-mice spinal cords and SMN-siRNA treated NSC34-cells. Transcript levels of SMA-mice and control animals were measured at P1, P5 and P8. NSC34-cells were either transfected with SMN- or control scrambled-siRNA in four independent experiments (n = 4) with three replicates in each group. The knockdown for each experiment was monitored by western-blot analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031202#pone-0031202-g002" target="_blank">Fig. 3</a>). The fold-changes were calculated in comparison to each corresponding control group. Fold-changes of SMA-mice spinal cords were calculated compared to transcript levels of control mice of the same age. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031202#s2" target="_blank">Results</a> of SMN siRNA treated cells were calculated compared to scrambled RNA transfected cells of the same experiment. SMA-mice transcript levels were tested against control animals by a Mann-Whitney test (n≥5, *p<0.05, **p<0.01). mRNA-levels of SMN siRNA transfected cells were tested against scrambled siRNA transfected cells by repeated measurements two way ANOVA (n = 4, **p = 0.0046). Bars for fold-changes represent means with standard error of mean (SEM).</p

Western blot analysis of NSC34-cells after SMN-knockdown.

<p>(<b>A</b>) Phosphorylation of FGF-downstream targets Akt and ERK was analyzed in siRNA treated (si) and scrambled siRNA (scr) transfected cells. Phospho-antibodies against Akt (pAkt, S473), and ERK1/2 (pERK, T202,T204) were used to quantify changes in phosphorylation levels compared to non-phosphorylated Akt, ERK. Four independent experiments with three replications were performed. (<b>B</b>) Densitometrical measurements of SMN-bands normalized by α-tubulin showed an efficient knockdown of 20±5% in comparison to control-siRNA-transfected cells. (<b>C</b>) pAkt normalized to non-phosphorylated Akt was upregulated by a factor of 2.8±0.7. (<b>D</b>) pERK normalized to non-phospho-ERK was upregulated by a factor of 2.1±0.3. (<b>E</b>) The functional link between FGFR-1 signaling and ERK-hyperphosphorylation was analyzed by application of the specific FGFR-1 inhibitor PD173074 (50 µM). Additionally, FGF-2 was added to the medium in a final concentration of 50 ng/ml. (<b>F</b>) Densitometrical measurements of pERK normalized to non-phospho ERK revealed an upregulation of 10.6±3.1 fold in scrambled siRNA transfected cells under FGF-2 incubation compared to scrambled siRNA transfected control conditions. When transfected with SMN siRNA, the pERK level rises up to 23.7±0.6 fold change. These differences disappeared after PD173074 incubation. Bars and values represent means with standard error of mean (SEM). Significance was tested via repeated measurements two-way ANOVA (B, C, D, n = 4, ***p<0.0001, **p<0.01, *p<0.05) and paired ratio t-test (F, n = 6 for control conditions and n = 3 for remaining conditions, **p<0.01, *p<0.05).</p

Regulation of neuronal differentiation by proteins associated with nuclear bodies.

Nuclear bodies are large sub-nuclear structures composed of RNA and protein molecules. The Survival of Motor Neuron (SMN) protein localizes to Cajal bodies (CBs) and nuclear gems. Diminished cellular concentration of SMN is associated with the neurodegenerative disease Spinal Muscular Atrophy (SMA). How nuclear body architecture and its structural components influence neuronal differentiation remains elusive. In this study, we analyzed the effects of SMN and two of its interaction partners in cellular models of neuronal differentiation. The nuclear 23 kDa isoform of Fibroblast Growth Factor - 2 (FGF-2(23)) is one of these interacting proteins - and was previously observed to influence nuclear bodies by destabilizing nuclear gems and mobilizing SMN from Cajal bodies (CBs). Here we demonstrate that FGF-2(23) blocks SMN-promoted neurite outgrowth, and also show that SMN disrupts FGF-2(23)-dependent transcription. Our results indicate that FGF-2(23) and SMN form an inactive complex that interferes with neuronal differentiation by mutually antagonizing nuclear functions. Coilin is another nuclear SMN binding partner and a marker protein for Cajal bodies (CBs). In addition, coilin is essential for CB function in maturation of small nuclear ribonucleoprotein particles (snRNPs). The role of coilin outside of Cajal bodies and its putative impacts in tissue differentiation are poorly defined. The present study shows that protein levels of nucleoplasmic coilin outside of CBs decrease during neuronal differentiation. Overexpression of coilin has an inhibitory effect on neurite outgrowth. Furthermore, we find that nucleoplasmic coilin inhibits neurite outgrowth independent of SMN binding revealing a new function for coilin in neuronal differentiation

Metalloprotease-Mediated Cleavage Of Plexind1 And Its Sequestration To Actin Rods In The Motoneuron Disease Spinal Muscular Atrophy (Sma)

Cytoskeletal rearrangement during axon growth is mediated by guidance receptors and their ligands which act either as repellent, attractant or both. Regulation of the actin cytoskeleton is disturbed in Spinal Muscular Atrophy (SMA), a devastating neurodegenerative disease affecting mainly motoneurons, but receptor-ligand interactions leading to the dysregulation causing SMA are poorly understood. In this study, we analysed the role of the guidance receptor PlexinD1 in SMA pathogenesis. We showed that PlexinD1 is cleaved by metalloproteases in SMA and that this cleavage switches its function from an attractant to repellent. Moreover, we found that the PlexinD1 cleavage product binds to actin rods, pathological aggregate-like structures which had so far been described for age-related neurodegenerative diseases. Our data suggest a novel disease mechanism for SMA involving formation of actin rods as a molecular sink for a cleaved PlexinD1 fragment leading to dysregulation of receptor signaling.WoSScopu